Natural products and their derivatives have been rich sources for drug discovery; it has been estimated that more than 60% of the drugs that are in the market derive from natural sources. However, during the last three decades, research aimed at exploiting natural products for drug discovery has seriously declined, in part due to our limite capability in new natural product discovery and analogue production. Innovative technology for natural product purification, quantification, overproduction, and diversification is urgently neede to make a paradigm shift in the field of natural product research and to promote natural product-based drug discovery and development. As tagging natural products can address these challenging aspects of natural product research, this proposal seeks to develop a novel strategy that overcomes the limitations of known methods for tagging natural products with a bioorthogonal functionality. To this end, we will explore the unusual synthetic biology that living systems offer for bioorthogonal functionalities and develop general platforms for the in situ labeling of natural products, particularly complex natural products that are difficult to be synthesized or derivatized chemically. The success of this strategy will directly produce orthogonally functionalized natural product analogues that can be subjected to facile chemical modification for drug screening. More importantly, tagging natural products with a unique chemical handle will enable the visualization, enrichment, quantification, and mode of action study of natural products through bioorthogonal chemistry, and thus have a profound impact on our ability to address challenging questions in natural product biosynthesis, biology, and pharmacology. In addition, a novel natural product-based, quantitative, high-throughput screening method based on this tagging strategy is proposed here as a useful tool for evolving natural product biosynthetic enzymes that can efficiently function in specific pathways to produce natural product analogues in high titers. This screening method will be leveraged to understand and engineer the biosynthesis of several important families of natural products for new antibiotic and anticancer drug discovery. The proposed interdisciplinary project bridges multiple innovations in natural product biosynthesis, protein engineering, and bioorthogonal chemistry, and if successful, will vastly expand the natural product research toolkit and have broad impact in enzymology, biosynthesis, and medicine. It is therefore uniquely suited to the New Innovator Award with respect to its significance, novelty, and the investigator's record of innovativeness and creativity. This project has minimal overlap with other efforts in the research group, while providing good synergy with various research projects and fitting perfectly in one of our main research themes: to harness the power of enzymes towards the biosynthesis of natural products with ultimate applications in advancing human health.